KR102654964B1 - Specific primer set of Ralstonia syzygii and method for detecting Ralstonia syzygii using the same - Google Patents

Abstract

The present invention provides a primer set for detecting Ralstonia syzygii , comprising an oligonucleotide primer of SEQ ID NO: 1 and an oligonucleotide primer of SEQ ID NO: 2; A composition containing the same; a kit containing it; and a method for detecting Ralstonia syzygii using the same.

Description

Specific primer set of Ralstonia syzygii and method for detecting Ralstonia syzygii using the same {Specific primer set of Ralstonia syzygii and method for detecting Ralstonia syzygii using the same}

The present invention provides a primer set for detecting Ralstonia syzygii , comprising an oligonucleotide primer of SEQ ID NO: 1 and an oligonucleotide primer of SEQ ID NO: 2; A composition containing the same; a kit containing it; and a method for detecting Ralstonia syzygii using the same.

Green blight (wilting) is a disease that causes rapid wilting (wilting) symptoms and is caused by the plant pathogenic bacterium Ralstonia solanacearum . Green blight is a soil disease distributed worldwide, causing serious economic losses in agricultural and horticultural production by damaging more than 200 species of plants, including major Solanaceae crops such as tomatoes, potatoes, peppers, eggplants, and tobacco. At first, the leaves near the growing point wilt, then recover for several days at night and on rainy days, then the entire body wilts, and eventually the entire plant turns green. If you cut the stem or cut the epidermis, you will see that the vascular tubes have turned brown, and if you cut the stem and float it in water, you will see a white-colored bacterial fluid flowing down. It is a high-temperature disease whose incidence increases in fields with high temperature and humidity, and occurs frequently in facility cultivation. Once it occurs, contact transmission is severe, so all diseased plants must be removed and the temperature and humidity must be lowered. However, it is very difficult to practice this on farms.

In general, Ralstonia solanacearum has high genetic diversity in nature and is called RSSC ( Ralstonia solanacearum species complex). After several existing classification systems for classifying RSSC, currently Ralstonia solanacearum (combination of Phylotype IIa and IIb), Ralstonia pseudosolanacearum (Phylotype I) and III), Ralstonia syzygii (Phylotype IV).

Xiaoman She et al., "Identification and Genetic Characterization of Ralstonia solanacearum Species Complex Isolates from Cucurbita maxima in China" Frontiers in Plant Science, vol.8, October 2017, Ralstonia solanacearum from pumpkin ( Cucurbita maxima ). ( Ralstonia solanacearum ) Some primer sets for detecting Phylotype I-IV strains are disclosed.

However, the current technology detects Ralstonia syzygii ( Ralstonia solanacearum Phylotype IV), the bacteria that causes foot blight, directly from the sample, and also detects and diagnoses Ralstonia bacteria that are genetically close and have similar symptoms. There is difficulty in

For the diagnosis of pathogenic microorganisms, real-time PCR using PCR, SYBR Green or TaqMan probes, and LAMP (Loop Mediated Isothermal Amplification) technologies have been developed and used, and conversion to a high-throughput diagnostic system is being used. Biomarker development using real-time PCR has been developed to obtain rapid and accurate diagnostic results.

However, no primer set specific to the Ralstonia syzygii species and suitable for real-time PCR has been disclosed to date.

Accordingly, the present invention can detect Ralstonia syzygii ( Ralstonia solanacearum Phylotype IV), the causative bacterium of rose blight, with high specificity and sensitivity, and is suitable for real-time PCR to extract DNA from the specimen. The technical task is to provide a primer set that can confirm infection and directly quantify (density measurement) without extraction.

The present inventors registered the primer set in Genbank (www.ncbi.nlm.nih.gov/) of the National Center for Biotechnology Information (NCBI) to produce a set of primers for specifically detecting Ralstonia syzygii. The specificity of the hypothetical protein gene of Ralstonia syzygii NCPPB3727 (GenBank accession NZ_JACWNE010000002.1, 228797-229180, protein ID WP_172840133.1) was confirmed through blastn and blastx programs.

Therefore, based on the hypothetical protein gene, a new primer set was created to amplify a 208bp fragment from Ralstonia syzygii , and using this, PCR and real-time PCR amplification tests were performed on several microbial strains. As a result of performing this, it was confirmed that the primer set produced an amplification product specifically only in Ralstonia syzygii , thereby completing the present invention.

Based on the above, the present invention provides a primer set for detecting Ralstonia syzygii , comprising the oligonucleotide primer of SEQ ID NO: 1 and the oligonucleotide primer of SEQ ID NO: 2.

The present invention also provides a composition for detecting Ralstonia syzygii , comprising a primer set represented by the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

The present invention also provides a kit for detecting Ralstonia syzygii, which includes a primer set represented by the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

In addition, the present invention includes the steps of (i) performing a DNA amplification reaction on a sample suspected of being infected with Ralstonia syzygii using a primer set represented by the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2; and (ii) detecting a DNA amplification product. It provides a Ralstonia syzygii detection method.

In addition, the present invention includes the steps of performing real-time PCR on a sample suspected of Ralstonia syzygii infection using a primer set represented by the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2; and analyzing the results of gene amplification by real-time PCR .

Using the primer set according to the present invention, Ralstonia syzygii can be specifically detected with excellent efficiency in a short period of time.

In particular, the primer set according to the present invention has a high detection sensitivity for Ralstonia syzygii and can be detected with excellent efficiency even when the amount of sample is small.

In addition, real-time PCR using the primer set according to the present invention can directly confirm infection by Ralstonia syzygii without DNA extraction from the sample, and can quantitatively analyze the pathogen entity to obtain a sample of the pathogen. You can check your density.

Figures 1A and 1B show the results of blasting the amplified region of the Ralstonia syzygii strain with the primer set according to the present invention. Figure 1A shows a list of all strains to be searched, and Figure 1B shows the corresponding The matching part of the strain is shown in the picture.
Figure 2 shows the results of electrophoresis after PCR amplification of genomic DNA of several bacterial strains, including Ralstonia syzygii , using the primer set of the present invention.
Lane M is 1 kb DNA plus ladder (Gibco BRL) as a size marker, lane 1 corresponds to Ralstonia syzygii strain, and lanes 2 to 22 are other Ralstonia species strains and Pseudomonas ( Pseudomonas ), Pectobacterium , Dickeya , Pantoea , and Xanthomonas (see Table 1), and lane 23 is a negative control (distilled water). . Specifically, lanes 2 to 22 are as follows: Lane 2: Ralstonia pseudosolanacearum ; Lane 3: Ralstonia pseudosolanacearum ; Lane 4: Ralstonia mannitolilytica ; Lane 5: Ralstonia pickettii ; Lane 6: Burkholderia gladioli ; Lane 7: Pseudomonas syringae pv. tomato ; Lane 8: Pseudomonas putida ; Lane 9: Pseudomonas glycinea ; Lane 10: Pseudomonas tabaci ; Lane 11: Pseudomonas fluorescens ; Lane 12: Pectobacterium carotovorum subsp. carotovorum ; Lane 13: Pectobacterium carotovorum subsp. carotovorum ; Lane 14: Pectobacterium carotovorum subsp. odoriferum ; Lane 15: Pectobacterium carotovorum subsp. brasilense ; Lane 16: Pectobacterium wasabiae ; Lane 17: Pectobacterium atrosepticum ; Lane 18: Dickeya chrysanthemi ; Lane 19: Pantoea agglomerans ; Lane 20: Pantoea stewartii ; Lane 21: Pantoea ananatis ; Lane 22: Xanthomonas axonopodis .
Figure 3 shows the results of real-time PCR on genomic DNA of several bacterial strains, including Ralstonia syzygii , using the primer set of the present invention. From the top, the amplification graph (top), dissolution curve graph (middle), and dissolution peak graph (bottom) are shown. Lane 1 corresponds to the Ralstonia syzygii strain, and lanes 2 to 22 correspond to other Ralstonia species strains and Pseudomonas , Pectobacterium , and Dickeya . , Pantoea , and Xanthomonas (see Table 1), and lane 23 corresponds to the negative control. The dissolution peak graph (bottom) shows the amplified product at a dissolution temperature (Tm) of approximately 85°C.
Figure 4 shows the results of real-time PCR analysis of 10-fold serial dilutions of cloned DNA, genomic DNA, and cell suspension of Ralstonia syzygii . Standard curves were generated from the threshold cycle (Ct) (also called “crossing point”) of Ralstonia syzygii TS3175 standard dilution. All curves showed R ² >0.99. (A) corresponds to replicated DNA, (B) to genomic DNA, and (C) to bacterial cell suspension.
Figure 5 shows the detection and density change of the pathogen in the rhizosphere using the primer set of the present invention after inoculating Ralstonia syzygii TS3175 into a tomato root sample using real-time PCR technique. It shows one result.

The present inventors registered the primer set in Genbank (www.ncbi.nlm.nih.gov/) of the National Center for Biotechnology Information (NCBI) to produce a set of primers for specifically detecting Ralstonia syzygii. The specificity of the hypothetical protein gene of Ralstonia syzygii NCPPB3727 (GenBank accession NZ_JACWNE010000002.1, 228797-229180, protein ID WP_172840133.1) was confirmed through blastn and blastx programs.

Accordingly, based on the hypothetical protein gene, a new primer set was created to amplify a 208bp fragment in Ralstonia syzygii . The base sequence of the primer set is as follows.

● RS02500-208F: 5'-GGA ATC CGC GCC TAA ACA ACT T-3' (22mer, SEQ ID NO: 1)

● RS02500-208R: 5'-CAA TGC GCT CGG GTC AAA ATC-3' (21mer, SEQ ID NO: 2)

The present inventors used the primer set to target other Ralstonia species, including Ralstonia syzygii, and Pseudomonas , Pectobacterium , Dickeya , and Panto. As a result of performing PCR and real-time PCR amplification tests on several microbial strains including Pantoea and Xanthomonas genera, the primer set was found to be specific only for Ralstonia syzygii. It was confirmed that an amplification product was produced.

Accordingly, in one aspect, the present invention relates to a primer set for detecting Ralstonia syzygii , comprising the oligonucleotide primer of SEQ ID NO: 1 and the oligonucleotide primer of SEQ ID NO: 2.

The RS02500-208F primer represented by the nucleotide sequence of SEQ ID NO: 1 is a forward primer, and the RS02500-208R primer represented by the nucleotide sequence of SEQ ID NO: 2 is a reverse primer.

When DNA is amplified using the primer set according to the present invention, bacteria other than Ralstonia syzygii are not amplified, and a band of about 208 bp is clearly detected, enabling accurate and easy detection.

As used herein, the term “primer” refers to a single-stranded oligonucleotide sequence complementary to the nucleic acid strand to be replicated, and can serve as a starting point for the synthesis of a primer extension product. When designing primers, there are various restrictions, such as the A, G, C, and T content ratio of the primers, prevention of primer complex (dimer) formation, and prohibition of repeating the same base sequence more than three times. In addition, in the individual PCR reaction conditions, the template ( template) Conditions such as amount of DNA, concentration of primer, concentration of dNTP, concentration of Mg ²⁺ , reaction temperature, reaction time, etc. must be appropriate.

The primer set according to the present invention may also include base sequences in which one or more bases are deleted, substituted, or added to the base sequences shown in SEQ ID NOs: 1 and 2. Additionally, the primer set according to the invention may contain additional features without changing the basic properties. That is, the base sequence can be modified using many means known in the art. Examples of such modifications include methylation, capping, substitution of a nucleotide with one or more homologs, and uncharged linkages such as phosphonates, phosphotriesters, phosphoroamidates, or carbamates, or phosphorothioates or phosphorodithiolates. Modification of nucleotides into charged linkages such as ates is possible. In addition, nucleic acids may contain one or more nucleases, toxins, antibodies, signal peptides, proteins such as poly L lysine, intercalating agents such as acridine or psoralen, chelating agents such as metals, radioactive metals, iron oxidizing metals, and alkylating agents. May have additional covalently linked residues.

Additionally, the base sequence of the primer set according to the present invention can be modified using a label that can directly or indirectly provide a detectable signal. The primer set may include a label that can be detected using spectroscopic, photochemical, biochemical, immunochemical or chemical means. Useful labels include ³² P, fluorescent dyes, electron dense reagents, enzymes (commonly used in ELISA), biotin or haptens, and proteins for which antisera or monoclonal antibodies are available.

The primer set according to the present invention is prepared by cloning of appropriate sequences, restriction enzyme digestion, phosphotriester method by Narang et al. (1979, Meth, Enzymol.68:90-99), diethylphosphatase method by Beaucage et al. It can be chemically synthesized using any other well-known method, including direct chemical synthesis methods such as the foramidite method (1981, Tetrahedron Lett. 22: 1859-1862), and the solid support method of U.S. Pat. No. 4,458,066. .

Methods for amplifying target nucleic acids include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, and transcription-based amplification system. ), strand displacement amplification or amplification via replicase or any other suitable method for amplifying nucleic acid molecules known in the art is possible.

Among these, “polymerase chain reaction (PCR)” is a method of amplifying a target nucleic acid from a set of primers that specifically bind to the target nucleic acid using a polymerase. The PCR of the present invention encompasses general (non-quantitative) PCR and quantitative PCR, and includes, for example, general PCR and real-time PCR. PCR is the best-known nucleic acid amplification method, and many modifications and applications have been developed. For example, touchdown PCR, hot start PCR, nested PCR, and booster PCR have been developed by modifying traditional PCR procedures to increase the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, and inverse polymerase chain reaction. IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. For more information about PCR, see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000).

Real-time PCR is a technology that monitors and analyzes the increase in PCR amplification products in real time. PCR reactions can be monitored by recording the fluorescence emission at each cycle during the exponential phase, when the increase in PCR product is proportional to the initial amount of target template. The higher the starting copy number of the nucleic acid target, the faster the fluorescence increase is observed and the lower the Ct value (threshold cycle). When the amount of PCR amplification product reaches an amount detectable by fluorescence, an amplification curve begins to occur, and the signal rises exponentially before reaching a plateau. The larger the initial amount of DNA, the fewer cycles it takes for the amount of amplification product to reach a detectable amount, so the amplification curve appears faster. Therefore, when a real-time PCR reaction is performed using a stepwise diluted standard sample, an amplification curve arranged at equal intervals in order of the initial DNA amount is obtained. Here, if the threshold is set at an appropriate point, the Ct value at the point where the threshold and the amplification curve intersect is calculated.

In one embodiment, the primer set according to the present invention may be used for PCR.

In another embodiment, the primer set according to the present invention may be used for real-time PCR.

In the present invention, the amplified target sequence can be labeled with a detectable labeling substance. In one embodiment, the labeling material may be a material that emits fluorescence, phosphorescence, or radioactivity, but is not limited thereto. When amplifying a target sequence, if the 5'-end of the primer is labeled with Cy-5 or Cy-3 and the amplification reaction is performed, the target sequence can be labeled with a detectable fluorescent labeling substance. In addition, when labeling using a radioactive material is performed when a radioactive isotope such as ³² P or ³⁵ S is added to the amplification reaction solution, radioactivity is incorporated into the amplification product as the amplification product is synthesized, and the amplification product can be radioactively labeled. .

In the present invention, detection of the amplification product may be performed through fluorescence measurement, DNA chip, gel electrophoresis, radioactivity measurement, or phosphorescence measurement, but is not limited thereto. As one of the methods for detecting amplification products, gel electrophoresis can be performed. Gel electrophoresis can use agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product.

Another aspect of the present invention relates to a composition for detecting Ralstonia syzygii , comprising a primer set represented by the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

Another aspect of the present invention relates to a kit for detecting Ralstonia syzygii, which includes a primer set represented by the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

In one embodiment, the kit may be a PCR kit.

In another embodiment, the kit may be a kit for real-time PCR.

In one embodiment, the kit may further include reagents and instructions for nucleic acid extraction, amplification, and product detection in addition to the primer set. For example, reagents for nucleic acid extraction, deoxynucleotides, reagents necessary for nucleic acid amplification reaction, heat-stable nucleic acid polymerase, buffer, dNTP, etc. may be included. The primer set and reagents for nucleic acid extraction, amplification, and product detection may be provided in a freeze-dried form in a plastic tube, and the nucleic acid may preferably be DNA.

Another aspect of the present invention is (i) performing a DNA amplification reaction on a sample suspected of being infected with Ralstonia syzygii using a primer set represented by the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2. ; and (ii) detecting a DNA amplification product. It relates to a method for detecting Ralstonia syzygii .

The sample in step (i) may include all those suspected of being infected with Ralstonia syzygii, such as these germs themselves or the leaves, branches, stems, and roots of plants expected to be infected with these germs. It may be a flower, etc.

In one embodiment, the DNA amplification reaction in step (i) may be real-time PCR. The primer set according to the present invention is suitable for real-time PCR, and can directly confirm infection with Ralstonia syzygii from the sample without DNA extraction, and quantitatively analyzes the pathogen entity to identify the pathogen. You can check the density within the sample.

In one embodiment, step (i) may further include the step of isolating genomic DNA from a sample suspected of being infected with Ralstonia syzygii. To isolate genomic DNA from a sample, methods known in the art can be used, for example, the CTAB method can be used, or the Wizard kit (Promega) can be used.

In one embodiment, in step (i), when genomic DNA is isolated from a sample suspected of being infected with Ralstonia syzygii , the DNA amplification reaction may be performed by PCR. The PCR reaction can be performed using a PCR reaction mixture containing various components known in the art required for the PCR reaction. The PCR reaction mixture contains an appropriate amount of DNA polymerase, dNTP, PCR buffer solution, and water in addition to genomic DNA and the primer pair provided in the present invention.

Detection of the DNA amplification product in step (ii) may be performed using a DNA chip, electrophoresis, radioactivity measurement, fluorescence measurement, or phosphorescence measurement, through which it can be confirmed whether the target gene has been amplified. Gel electrophoresis can be performed as one of the methods for detecting amplification products. Gel electrophoresis can use agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product.

In one embodiment, the detection of the DNA amplification product in step (ii) may be performed by electrophoresis, and at this time, the amplification of the target gene is determined by the 208bp size of the hypothetical protein gene of Ralstonia syzygii. This can be confirmed by whether or not the fragment is amplified.

Another aspect of the present invention includes performing real-time PCR on a sample suspected of being infected with Ralstonia syzygii using a primer set represented by the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2; It relates to a method for diagnosing Ralstonia syzygii infection, comprising analyzing the results of gene amplification by real-time PCR.

In one embodiment, the diagnostic method is characterized in that real-time PCR is performed on the sample itself without isolating genomic DNA from a sample suspected of Ralstonia syzygii infection.

The real-time PCR diagnostic method according to the present invention enables the detection of the bacteria directly from samples suspected of Ralstonia syzygii infection without DNA extraction. In addition, the real-time PCR diagnostic method can quantitatively analyze Ralstonia syzygii individuals from a sample to confirm the density of the pathogen in the sample.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example

Example 1: Bacterial strain culture and total DNA extraction

(1) Bacterial strains and culture conditions

Other microorganisms, including Ralstonia syzygii , used in the present invention were distributed by the Belgian Co-ordinated Collections of Microorganisms (BCCM ^TM ) and Korean Agricultural Culture Collection, Republic of Korea (KACC) in Belgium. The sources are summarized in Table 1. Ralstonia , Pseudomonas , Pectobacterium , Dickeya , Pantoea , and Xanthomonas strains were each cultured in TSA medium at 27°C for two days. .

[Table 1] Bacterial strains used in PCR specificity test

KACC: Korean Agricultural Culture Collection, Republic of Korea

LMG: The Belgian Co-ordinated Collections of Microorganisms (BCCM ^TM ), Belgium

^a Superscript "T" indicates type strain.

^b ND, not determined.

(2) Extraction of total DNA

Total DNA of the cultured strain was extracted using a genomic DNA extraction kit (Wizard ^® Genomic DNA Purification Kit) from Promega (Madison, USA).

Example 2: Preparation of a specific primer set

(1) Analysis using blastn program to search for specific nucleotide sequence information

Ralstonia registered in Genbank (www.ncbi.nlm.nih.gov/) of the National Center for Biotechnology Information (NCBI) for the production of a primer set to specifically detect Ralstonia syzygii. The specificity of the hypothetical protein gene of the genomic information of Ralstonia syzygii NCPPB3727 (GenBank accession NZ_JACWNE010000002.1, 228797-229180, protein ID WP_172840133.1) was confirmed through blastn and blastx programs (Figure 1).

Figures 1a and 1b show the results of blasting the amplified region of the Ralstonia syzygii strain with the primer set according to the present invention, Figure 1a shows a list of all strains to be searched, and Figure 1b shows The matching region of the strain is shown graphically, showing high similarity (red bars) in most gene sequences only in the Ralstonia syzygii genome, while other Ralstonia species show low similarity. (green bar) could be confirmed.

(2) Production of specific primers

Primer sets RS02500-208F and RS02500-208R were created to amplify a fragment of 208bp in size from Ralstonia syzygii . The base sequence of the primer is as follows.

● RS02500-208F: 5'- GGA ATC CGC GCC TAA ACA ACT T-3' (22mer, SEQ ID NO: 1)

● RS02500-208R: 5'-CAA TGC GCT CGG GTC AAA ATC-3' (21mer, SEQ ID NO: 2)

Example 3: PCR amplification reaction

PCR amplification reaction was performed using a PTC-200 ^TM thermocycler (MJ research, Watertown, mass), and each reaction ^consisted of 1 The PCR was performed in a total volume of 25 μl using a PCR mixture containing 1.25 units of enzyme (Promega Madison, WI, USA). The amount of genomic DNA of some microorganisms added to the PCR mixture was approximately 25 ng. The reaction was denatured at 95°C for 5 minutes, repeated 35 times at 95°C for 60 seconds, 63°C for 60 seconds, and 72°C for 60 seconds, and then reacted at 72°C for 10 minutes. After the amplification reaction, 10 μl of the PCR product was stained with LoadingStar dye (DYNEBIO, Republic of Korea), electrophoresed on a 1.5% concentration agarose gel, and the amplified DNA band was confirmed in a UV transilluminator. . The electrophoresis results are shown in Figure 2.

In Figure 2, lane 1 corresponds to the Ralstonia syzygii strain, and lanes 2 to 22 correspond to other Ralstonia species strains and Pseudomonas , Pectobacterium , and Dickey. ( Dickeya ), Pantoea , and Xanthomonas (see Table 1), and lane 23 corresponds to the negative control. As shown in lane 1 of Figure 2, it was confirmed that a 208bp nucleic acid fragment was specifically amplified only in Ralstonia syzygii . No bands were observed in other lanes 2 to 23.

As such, the primer set of SEQ ID NO: 1 and SEQ ID NO: 2 specifically amplifies only Ralstonia syzygii , and Ralstonia pseudosolanacearum , Ralstonia mannitolilytica , Ralstonia pickettii , etc., which are genetically close to Ralstonia syzygii and have similar symptoms. It was found that other Ralstonia species were not amplified. In addition, the primer set of SEQ ID NO: 1 and SEQ ID NO: 2 did not amplify strains of the genera Pseudomonas , Pectobacterium , Dickeya , Pantoea , and Xanthomonas . Therefore, it can be seen that the primer sets of SEQ ID NO: 1 and SEQ ID NO: 2 are specific only to Ralstonia syzygii species.

Example 4: Real-time PCR specificity analysis

Real-time PCR amplification reaction was performed using the CFX96 Real-time PCR system (Bio-Rad laboratories, Inc., USA), and each reaction was performed using TB green ^TM Ex Taq ^TM Ⅱ (Tli RNaseH Plus, 2X) (TAKARA Bio , Inc., Japan) and RS02500-208F/R primers were performed in a total volume of 20 μl with a real-time PCR mixture containing 5 pM each.

The amount of microbial genomic DNA added to the real-time PCR mixture was approximately 5 ng. The reaction was denatured at 95°C for 30 seconds, repeated 40 times at 95°C for 5 seconds and 63°C for 30 seconds, then reacted at 95°C for 15 seconds, and then dissolved at 65 to 95°C for the melting curve and melting peak. The temperature was increased by 0.5°C every 5 seconds. Real-time PCR results are shown in Figure 3.

Figure 3 shows the amplification graph (top), dissolution curve graph (middle), and dissolution peak graph (bottom) from the top, with lane 1 corresponding to the Ralstonia syzygii strain, and lanes 2 to 22. Corresponds to strains of other Ralstonia species and strains of the genera Pseudomonas , Pectobacterium , Dickeya , Pantoea , and Xanthomonas (see Table 1 ), lane 23 corresponds to the negative control.

The dissolution peak graph (bottom) shows the amplified product at a dissolution temperature (Tm) of approximately 85°C. As shown in Figure 3, the amplification reaction occurred only in the Ralstonia syzygii strain, and a dissolution peak was confirmed at a specific temperature (85°C).

Example 5: Real-time PCR sensitivity analysis

Real-time PCR amplification reaction was performed using the CFX96 Real-time PCR system (Bio-Rad laboratories, Inc., USA), and each reaction consisted of SYBR premix Ex Taq (2x) (TAKARA Bio, Inc., Japan) and The real-time PCR mixture containing 5 pM of each RS02500-208F/R primer was performed in a total volume of 20 μl. Ralstonia syzygii TS3175 genomic DNA was diluted 10 times from 5 ng, and the amount of cloned DNA was diluted 10 times from 100 pg. Then, the microbial suspension was adjusted to OD ₆₀₀ to 0.1, then diluted 10 times and added to the real-time PCR mixture. Real-time PCR amplification reaction is denatured at 95℃ for 30 seconds, repeated 40 times at 95℃ for 5 seconds and 65℃ for 30 seconds, then reacted at 95℃ for 15 seconds, and then incubated at 65~95℃ for dissolution curve and dissolution peak. It was increased by 0.5°C every second. Real-time PCR sensitivity analysis results are shown in Figure 4 and Table 2 below.

[Table 2] Ralstonia sizikii measured by real-time PCR analysis ( Ralstonia syzygii ) Average Ct endpoint fluorescence of 10-fold serial dilutions of cloned DNA, genomic DNA, and cell suspension of TS3175.

The standard curve in Figure 4 was generated from the threshold cycle (Ct) (also called "crossing point") of the standard dilution of Ralstonia syzygii TS3175. All curves showed R ² >0.99. The R ² value and E value of the standard curve in FIG. 4 are considered to be within an appropriate range, which can be confirmed as valid data. As shown in Figure 4 and Table 2 above, Ralstonia syzygii at 100 ag for replicated DNA, at 500 fg for genomic DNA, and at OD ₆₀₀ = 0.0001 for bacterial cell suspension. It was confirmed that stable quantitative detection was possible.

From the above results, it was found that the RS02500-208F/R primer set of the present invention can detect Ralstonia syzygii with high sensitivity in real-time PCR.

Example 6: Real-time PCR amplification using tomato root samples

For quantitative analysis using real-time PCR at each hour after inoculation of tomato root samples with Ralstonia syzygii TS3175, Moneymaker, a susceptible variety, was grown in a greenhouse. Ralstonia syzygii TS3175 was cultured in TSB medium with shaking at 27°C for 2 days, and 5 ml of each was inoculated at an OD ₆₀₀ value of 1.0. Roots were collected for each variety at intervals of 4, 7, and 10 days, including day 0 before inoculation. After removing as much of the soil attached to the roots as possible as a pretreatment, it was transferred to a 50 ml tube, 30 ml of sterilized water was added, and strongly suspended using a voltexer. After immersion for 10 minutes, the suspension was used. Put 1 ml of the suspension in a 1.5 ml tube, centrifuge at 13,000 rpm for 10 minutes, and then use the pellet collected below to extract total DNA using the Fast DNA ^® spin kit for soil from MPbio (OH, USA). time PCR was performed.

Real-time PCR amplification reaction was performed using the CFX96 real-time PCR system (Bio-Rad laboratories, Inc., USA), and each reaction contained 5 pM each of SYBR Premix Ex Taq (2x) and RS02500-208F/R primers. It was performed in a total volume of 20 μl with the SYBR-Green PCR mixture containing. The total amount of DNA from the tomato root sample added to the PCR mixture was approximately 5 ng. The reaction was denatured at 95°C for 30 seconds, repeated 40 times at 95°C for 5 seconds, and 63°C for 30 seconds, then reacted at 95°C for 15 seconds, then reacted for 5 seconds at 0.5°C intervals starting from 65°C, and then increased to 95°C. While doing so, the dissolution curve and dissolution peak were performed. As a result, specific curves and specific peaks were confirmed in all diseased tomato root samples, and the detection and density changes of the corresponding pathogens in the rhizosphere are shown in Figure 5.

As shown in Figure 5, Ralstonia syzygii TS3175 was not detected before inoculation, and on the 4th day of inoculation, the Ct value (threshold cycle) was detected at about 26.98, maintained until the 7th day, and the Ct value decreased on the 10th day. It was confirmed that it increased to about 25.43.

In conclusion, the RS02500-208F/R primers prepared from the specific DNA fragment of the hypothetical protein gene of Ralstonia syzygii are PCR primers for specific detection of Ralstonia syzygii. Alternatively, it can be seen that it can be used as a real-time PCR primer, and it has been confirmed that it can be applied to qualitative and quantitative analysis and monitoring of density changes of Ralstonia syzygii in the tomato rhizosphere.

<110> Republic of Korea (Management: Rural Development Administration) <120> Specific primer set of Ralstonia syzygii and method for detecting Ralstonia syzygii using the same <130>PD21-267 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RS02500-208F forward primer <400> 1 ggaatccgcg cctaaacaac tt 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> RS02500-208R reverse primer <400> 2 caatgcgctc gggtcaaaat c 21

Claims (15)

A primer set for detecting Ralstonia syzygii , comprising the oligonucleotide primer of SEQ ID NO: 1 and the oligonucleotide primer of SEQ ID NO: 2. The primer set of claim 1, wherein the primer set is used in polymerase chain reaction (PCR). The primer set according to claim 1, wherein the primer set is used for real-time PCR. A composition for detecting Ralstonia syzygii, comprising the primer set of any one of claims 1 to 3. A kit for detecting Ralstonia syzygii, comprising the primer set of any one of claims 1 to 3. The kit according to claim 5, wherein the kit is for PCR (polymerase chain reaction). The kit according to claim 5, wherein the kit is for real-time PCR. (i) performing a DNA amplification reaction on a sample suspected of being infected with Ralstonia syzygii using the primer set of any one of claims 1 to 3; and
(ii) detecting the DNA amplification product,
Ralstonia syzygii detection method. The method of claim 8, wherein the DNA amplification reaction in step (i) is real-time PCR. The method of claim 8, further comprising the step of isolating genomic DNA from a sample suspected of being infected with Ralstonia syzygii in step (i). The method of claim 10, wherein the DNA amplification reaction in step (i) is PCR. 9. The method according to claim 8, wherein the detection of the DNA amplification product in step (ii) is performed by electrophoresis. The method according to claim 12, wherein whether the target gene is amplified is confirmed through whether a fragment of 208bp in size is amplified. Performing real-time PCR on a sample suspected of being infected with Ralstonia syzygii using the primer set of any one of claims 1 to 3; And a method for diagnosing Ralstonia syzygii infection, comprising analyzing the results of gene amplification by real-time PCR. The method of claim 14, wherein real-time PCR is performed on the sample itself without isolating genomic DNA from a sample suspected of being infected with Ralstonia syzygii .